Evidence of a role for CutRS and actinorhodin in the secretion stress response in Streptomyces coelicolor M145

CutRS was the first two-component system to be identified in Streptomyces species and is highly conserved in this genus. It was reported >25 years ago that deletion of cutRS increases the production of the antibiotic actinorhodin in Streptomyces coelicolor . However, despite this early work, the function of CutRS has remained enigmatic until now. Here we show that deletion of cutRS upregulates the production of the actinorhodin biosynthetic enzymes up to 300-fold, explaining the increase in actinorhodin production. However, while ChIP-seq identified 85 CutR binding sites in S. coelicolor none of these are in the actinorhodin biosynthetic gene cluster, meaning the effect is indirect. The directly regulated CutR targets identified in this study are implicated in extracellular protein folding, including two of the four highly conserved HtrA-family foldases: HtrA3 and HtrB, and a putative VKOR enzyme, which is predicted to recycle DsbA following its catalysis of disulphide bond formation in secreted proteins. Thus, we tentatively propose a role for CutRS in sensing and responding to protein misfolding outside the cell. Since actinorhodin can oxidise cysteine residues and induce disulphide bond formation in proteins, its over production in the ∆cutRS mutant may be a response to protein misfolding on the extracellular face of the membrane.


INTRODUCTION
Streptomyces species are ubiquitous soil bacteria that play important roles in the turnover of organic material in the soil and form beneficial interactions with plants and insects [1,2]. They are studied due to their complex developmental life cycles, which include hyphal growth, sporulation, and exploration [3], and because they make a diverse array of bioactive specialized metabolites, many of which are used clinically as antibiotics [4]. Understanding the control of the complex Streptomyces life cycle in response to nutritional and other environmental cues is a major challenge and is important because the progression of the life cycle into sporulation is linked to the production of antibiotics [5]. With 97 % of their specialized metabolite biosynthetic gene clusters (BGCs) yet to be matched to molecules, understanding the environmental signals and signalling pathways that control the expression of these cryptic gene clusters is expected to lead to the discovery of new molecules, including new antibiotics [6].
One of the major ways in which Streptomyces bacteria sense and respond to their environment is via two-component systems (TCS), which consist of a sensor histidine kinase (SK), which usually spans the cell membrane, and a cytoplasmic response OPEN ACCESS regulator (RR) that typically modulates target gene expression [7]. The SK senses a signal outside the cell, autophosphorylates its cytoplasmic kinase domain, and the RR then catalyses the transfer of that phosphate to its own receiver domain to switch on its DNA binding activity. Many of the TCS that have been characterized in the genus Streptomyces have been shown to either directly or indirectly affect antibiotic production, but relatively few systems have been characterized in detail [8]. We recently compared 93 complete Streptomyces genome sequences and identified 15 conserved TCS encoded by these bacteria, one of which is called CutRS [9]. This was the first TCS to be identified in the genus Streptomyces >25 years ago and the genes (a complete cutR and partial cutS) were first cloned and sequenced because a mutation in cutR was thought to suppress a mutation in melC1, the gene required for the assembly of the copper containing tyrosinase MelC2. The TCS was named CutRS because it was thought to be involved in regulating copper metabolism [10]. However, a later study, which cloned and sequenced the entire cutRS operon and compared it to the cutRS genes in the melC1 suppressor strain, revealed there were no mutations and they concluded that the suppressor lies outside of cutRS [11]. This work also reported that deletion of cutRS in Streptomyces coelicolor or Streptomyces lividans increases production of the blue-pigmented antibiotic actinorhodin and the authors concluded that CutR represses actinorhodin biosynthesis. Despite this pioneering early work, the function of CutRS, its target genes, and its role in controlling antibiotic production remain unknown.
In this work we set out to investigate the role of CutRS in controlling actinorhodin production in S. coelicolor M145. We confirmed that deletion of cutRS increases actinorhodin production in this species and report that an S. coelicolor ∆cutRS mutant can be complemented in trans by its own cutRS operon and those from the distantly related species Streptomyces formicae [12] and Streptomyces venezuelae [13], supporting the hypothesis that CutRS structure and function are highly conserved in this genus. We then used ChIP-seq to identify the conserved CutR regulon in S. coelicolor using both the native cutRS (SccutRS) and the heterologously expressed S. venezuelae cutRS (SvcutRS) genes. Intriguingly, we found that DNA binding by ScCutR and SvCutR in S. coelicolor is dependent on the addition of glucose to the growth medium and we identified 16 binding sites that are shared by both CutR proteins on the S. coelicolor genome. Through quantitative and comparative tandem-mass tagging (TMT) proteomics we show that CutR directly controls the production of S. coelicolor HtrA3 and HtrB, which are two of the four conserved HtrA (high temperature requirement) proteins in Streptomyces species. This family of proteins typically act as chaperones and quality control proteases for secreted proteins and their production is controlled in response to secretion and/or cell envelope stress [14]. CutR also directly controls the production of a putative vitamin K epoxide reductase (VKOR) enzyme SCO1507, a member of group of enzymes that perform the same function as DsbB, i.e. they recycle DsbA following its catalysis of disulphide bond formation in secreted proteins [15]. Thus, we propose that CutRS has a role in the secretion stress response in S. coelicolor, which is triggered when proteins fold incorrectly following export through the general secretion pathway (Sec). Whilst the actinorhodin biosynthetic enzymes are increased up to 300-fold in the ∆cutRS mutant, the over-production of actinorhodin must be indirect because CutR does not bind within the actinorhodin BGC. Since actinorhodin is redox active and can oxidise cysteine residues [16], it is possible this specialized metabolite is produced to assist the folding of secreted proteins under times of stress.

Strains, plasmids and primers
All bacterial strains used in this work are listed in Table 1 and the plasmids used in this work are listed in Table 2. Those that were generated for this work were constructed as follows: DNA fragments containing 25-35 nucleotide overlapping regions were assembled into digested DNA vectors using the exonuclease-based Gibson Assembly (NEB). A standard 3 : 1 ratio of insert to vector was used and assembly performed at 50 °C for 1 h in a thermocycler. Primers used in this work are listed in Table 3.

Impact statement
It is important for bacteria to sense their environment in order to survive in dynamic and changing niches. Two-component systems (TCS), typically consisting of a membrane-bound histidine kinase and a cognate response regulator, are a common example of these types of sensors. Streptomyces spp. are Gram-positive bacteria with complex life cycles, occupying diverse environmental niches and with the ability to produce a wide array of specialized metabolites, including antibiotics that are useful to humans. CutRS was the first two-component system to be identified in the genus Streptomyces and deletion of the cutRS genes from the S. coelicolor genome led to abnormally high levels of the redox active antibiotic actinorhodin being produced. In this work we have further characterized the mechanisms that underpin this relationship. It was shown, by identifying the genome locations where CutR (the response regulator) binds, that the direct effect of this TCS is not on the actinorhodin biosynthetic gene cluster but rather genes involved in secretion stress responses. This is interesting as it suggests that actinorhodin may be produced as a participant in the secretion stress response. Furthermore, our work highlights the importance of understanding the regulation of bacterial specialized metabolite production for the purposes of new molecule discovery and industrial production.

Growth media and conditions
The media used are listed in Table 4. The antibiotics used for selection of plasmid carriage were 50 µg ml −1 of apramycin and 50 µg ml −1 hygromycin as indicated by the resistance markers listed in Table 2. After conjugation, 25 µg ml −1 of nalidixic acid was used to kill E. coli and select for Streptomyces species.

CRISPR/Cas9 mediated deletion of cutRS in Streptomyces coelicolor
This methodology for scar-less gene deletion and genome editing in Streptomyces uses CRISPR/Cas9 as previously described [17]. Using CRISPy-web (https://crispy.secondarymetabolites.org, RRID:SCR_017970) a 20 nucleotide protospacer was designed to target the region of interest via a synthetic guide RNA (sgRNA). In total, 24 nucleotide primers were designed to construct this protospacer containing 5′ BbsI sticky ends. The pair of oligonucleotides were resuspended to 100 µM in deionised water (dH 2 O) before 5 µl of both were mixed in 90 µl 30 mM HEPES pH 7.8. The mixture was heated to 95 °C for 5 min before ramping to 4 °C at a rate of 0.1 °C per second. Golden gate [18] assembly was used to insert the annealed protospacer into pCRISPomyces-2 at the BbsI site and the resulting vector heat shocked into chemically competent E. coli Top10 (Invitrogen) before plating on selective LB containing IPTG and X-Gal. White colonies were picked into 10 ml selective LB for overnight incubation. Following plasmid preparation vectors were sequence confirmed and digested with XbaI. Gibson Assembly [19] was used to assemble two 1 kb PCR-amplified homology repair templates, matching regions adjacent to the target region, into the vector. The sequenceconfirmed final vector was moved into Streptomyces via E. coli ET12567/pUZ8002 conjugation and apramycin-resistant colonies were PCR-screened for desired mutations. Finally, the mutants were passaged for multiple generations at 37 °C to facilitate the loss of the temperature-sensitive pCRISPomyces-2.

Conjugation of plasmids to S. coelicolor via E. coli ET12567/pUZ8002
Single colonies of E. coli ET12567/pUZ8002 containing the desired target vector were picked from selective LB agar plates and incubated overnight in 10 ml selective LB broth, shaking at 220 r.p.m. The overnight culture was sub-cultured in 50 ml selective LB broth and grown to OD 600 0.6. The cultures were washed with 10 ml ice-cold LB broth twice by centrifugation to remove the  residual antibiotics and finally resuspended in 1 ml ice-cold LB broth. Then, 20-50 µl of Streptomyces spores were suspended in 500 µl 2xYT and heat shocked at 50 °C for 10 min. Overall, 500 µl of the E. coli suspension was combined with the spore suspension, mixed by inversion and pelleted at 13 000 r.p.m. for 2 min. The supernatant was removed, and the pellet resuspended in 150 µl residual supernatant. Serial dilutions were performed and plated on SFM agar +10 mM MgCl 2 . Plates were incubated at 30 °C for 16-20 h and subsequently overlaid with 1 ml sterile dH 2 O containing 1.25 mg of the selective antibiotic and 0.5 mg nalidixic acid. Once dried, the plates were incubated at 30 °C for 3-7 days until colonies appeared. Colonies were then re-streaked onto SFM agar containing selective antibiotics (including nalidixic acid) at least once before being plated for spore preparation [20,21].

Tandem-mass-tagging proteomics
S. coelicolor colonies were grown on cellophane covered DNA or DNAD agar plates as triplicate spots from 5 µl spores, all in duplicate. After 5 days at 30 °C the mycelium was scraped into a 15 ml Falcon tube and resuspended in 10 ml cell lysis buffer [50 mM TEAB buffer pH 8.0, 150 mM NaCl, 2 % SDS, EDTA-free protease inhibitor, PhosSTOP phosphatase inhibitor (Sigma Aldrich)]. The suspension was disrupted via French press three times before being boiled for 10 min. Samples were sonicated at 50 kHz four times for 20 s per cycle and then pelleted at 4 000 r.p.m. for 30 min. Protein concentration was determined using the BCA assay and 1 mg of protein from each sample transferred to a fresh 15 ml Falcon tube. Four volumes of methanol were added and vortexed thoroughly before one vol of chloroform was added and vortexed thoroughly. Three volumes dH 2 O was added, vortexed thoroughly, and spun for 10 min at 4 000 r.p.m. The upper layer was carefully discarded, four volumes of methanol were added and vortexed thoroughly. The samples were spun for 20 min at 4 000 r.p.m. before aspirating the supernatant.
Pellets from the protein extraction were washed with acetone and dissolved in 50-100 µl of 0.2 M EPPS buffer pH8 (Merck) with 2.5 % sodium deoxycholate (SDC; Merck). After quantification by BCA assay, 100 µg of protein was reduced and alkylated with 1,4-dithiothreitol and iodoacetamide and digested with trypsin according to standard procedures. After digestion, the SDC was precipitated by adjusting to 0.2 % TFA, and the clarified supernatant subjected to C18 solid phase extraction (SPE; OMIX tips; Agilent). TMT labelling was performed using a Thermo TMT16plex kit according to the manufacturer's instructions with slight modifications; the dried peptides were dissolved in 90 µl of 0.2 M EPPS buffer pH8/10 % acetonitrile, and 250 µg TMT16plex reagent dissolved in 22 µl of acetonitrile was added. Samples were assigned to the TMT channels in an order avoiding channel leakage between different samples, as detailed by [22].
After labelling, aliquots of 1.7 µl from each sample were combined and analysed on the mass spectrometer (detailed below) to check labelling efficiency and estimate total sample abundances. The sample aliquots were then combined correspondingly and desalted using a 50 mg C18 Sep-Pak cartridge (Waters). The eluted peptides were dissolved in 500 µl of 25 mM NH 4  The workflow started with spectrum recalibration. The database search was performed using the chimerys node (MSAID, Munich, Germany) with the inferys_2.1_fragmentation model, enzyme trypsin with one missed cleavage, 0.5 Da fragment tolerance, oxidation (M) as variable modification, carbamidomethyl (C) and TMT10plex (K, N-term) as static modifications. A parallel search was performed using the Comet node with the Comet_version 2019.01 rev. 0 parameter file. For Comet the precursor tolerance was set to 6 ppm, otherwise the same parameters as for chimerys were used. Matches were evaluated using Percolator based on q-values. Reporter ions were extracted using the most confident centroid with 20 ppm integration tolerance. The consensus workflow included the following parameters: only unique peptides (protein groups) for quantification, intensitybased abundance, channel correction values applied (TMT Lot VI306840), co-isolation/SPS matches thresholds 50 %/70 %, normalized chimerys Coefficient Threshold 0.8, normalization on total peptide abundances, protein abundance-based ratio calculation, missing values imputation by low abundance resampling, hypothesis testing by t-test (background based), adjusted P-value calculation by BH-method. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository [23] with the dataset identifier PXD040579.

Chromatin immuno-precipitation followed by sequencing (ChIP-Seq)
Streptomyces strains were inoculated onto cellophane covered DNA and DNAD agar plates as triplicate colonies using 5 µl spores for each and grown for 5 days at 30 °C. To cross-link proteins to DNA the cellophane discs were removed and submersed in Raw sequencing data were received as FASTQ files from Novogene and reads were aligned to the relevant reference genome. A normalized local enrichment was calculated along the chromosome by comparing the density of mapped reads in 30 nucleotide windows moving in steps of 15 nucleotides to the density of mapped reads in the 3000-nucleotide region surrounding the window.

qRT-PCR on htrA3 and htrB
To perform quantitative RT-PCR (qRT-PCR), S. coelicolor WT and ∆cutRS strains were grown on DNA or DNAD agar on top of sterilized cellophane discs at 30 °C for 9 days. Colonies were removed from the cellophanes kept in liquid nitrogen whilstcrushed using a sterile pestle and mortar on dry ice. Crushed samples were resuspended in 1 ml RLT Buffer (Qiagen) supplemented with 1 % 2-mercaptoethanol. This suspension was added to a QIA-shredder column (Qiagen) with the flowthrough transferred to a new 1.5 ml microfuge tube (leaving the pellet). Then, 700 µl acidic phenol:chloroform was added and incubated at room temperature for 3 minutes, and subsequently centrifuged at 13 000 r.p.m. for 20 min.

CutRS represses actinorhodin production in a glucose-dependent manner
We made a fresh ∆cutRS mutant in S. coelicolor M145 and confirmed that it over-produces the blue-pigmented antibiotic actinorhodin when grown on Difco nutrient agar supplemented with d-glucose (DNAD, Fig. 1). However, there is no obvious growth or developmental phenotype and the S. coelicolor ∆cutRS mutant does not visibly produce actinorhodin when grown on Difco nutrient agar without glucose (DNA, Fig. 1). This is surprising because glucose generally represses antibiotic production through glucose-mediated carbon catabolite repression [24]. Given the cutRS operon is highly conserved in Streptomyces genomes [9] we attempted to complement the S. coelicolor ∆cutRS mutant with the cutRS genes from S. coelicolor and from the more distantly related species S. venezuelae [13] and S. formicae [12]. In all cases, the introduction of these genes back into the mutant reversed the over-production of actinorhodin suggesting they are complementing the mutation (Fig. 1). The differences in colony morphology observed in Fig. 1 are likely due to the natural biological variation generally observed when culturing Streptomyces species.
Next, we compared the proteomes of wild-type S. coelicolor and the isogenic ∆cutRS strain using quantitative tandem-mass-tagged (TMT) proteomics to determine if the actinorhodin biosynthetic pathway is upregulated. The data detected 19 of the 22 of the proteins encoded by the actinorhodin BGC and they are all increased in abundance between 3.5-and 300-fold in the ∆cutRS mutant relative to the wild-type strain grown on DNAD. Three of the proteins encoded by the actinorhodin BGC were not detected in this experiment (Table 5). When the strains were grown without glucose 17 of the 19 proteins encoded by the actinorhodin BGC were upregulated between two-and tenfold in the ∆cutRS mutant relative to wild-type whereas levels of the ActAB actinorhodin transporter were lower in the mutant strain (Table 5). ActII-4, the cluster situated activator of this pathway, which activates the expression of the other genes in the BGC, is fivefold higher on DNAD and only threefold higher on DNA whereas the change for ActR, the cluster situated repressor, is less substantial, upregulated 3.6-fold on DNAD and 2.7-fold on DNA. However, glucose is not acting via CutRS (which are absent) and we cannot explain the repressive effect of CutRS on the actinorhodin biosynthesis pathway because CutR does not bind anywhere within the actinorhodin BGC as described in detail below.

Identifying CutR binding sites on the S. coelicolor genome
We complemented the ∆cutRS mutant with the SccutRS operon encoding wild-type CutS and C-terminally 3×Flag-tagged ScCutR (Fig. 1) and then performed ChIP-seq on the Flag-tagged and wild-type (negative control) strains using monoclonal anti-Flag antibodies. The strains were grown on DNA and DNAD plates for 3 days before the mycelium was cross-linked with formaldehyde and harvested for ChIP-seq analysis. For S. coelicolor grown without glucose (DNA), the sequence data show there were no significantly enriched targets (>twofold) in the Flag-tagged ScCutR strain relative to the wild-type, whereas in the presence of glucose (DNAD) there were 85 sites of enrichment in the Flag-tagged ScCutR strain (Table S1). Notably, ScCutR did not bind Fig. 1. S. coelicolor ∆cutRS over-produces the blue antibiotic actinorhodin on growth medium containing glucose and can be complemented by the cutRS genes from distantly related species. Agar plate grown cultures of, from left: [1] wild-type S. coelicolor M145 [2], S. coelicolor ∆cutRS [3], ∆cutRS containing pIJ10257 with the S. venezuelae cutRS operon under the control of the ermE* promoter (ermEp*) [4]; ∆cutRS containing pIJ10257 with the S. venezuelae cutRS operon under the control of ermEp* [5]; ∆cutRS containing pIJ10257 with the S. formicae cutRS operon under the control of the ermEp* [6]; ∆cutRS containing the S. venezuelae cutRS operon under the control of its native promoter and encoding a Flag-tagged CutR [7]; ∆cutRS containing pIJ10257 with the S. coelicolor cutRS operon under the control of its native promoter and encoding a Flag-tagged CutR [8]; ∆cutRS containing the empty pIJ10257 vector; The plates contain Difco nutrient agar without d-glucose (DNA) or with d-glucose (DNAD) as indicated.
upstream of any genes in the actinorhodin BGC suggesting that ScCutR repression of actinorhodin biosynthesis is indirect. Of the 85 direct target genes, 20 encode hypothetical proteins of unknown function, seven encode putative transcription factors, 38 encode predicted membrane or secreted proteins, 13 are related to primary or secondary metabolism and seven are implicated in protein synthesis. CutRS is highly conserved in the genus Streptomyces and the S. venezuelae cutRS genes complement the S. coelicolor ∆cutRS mutant so we repeated the ChIP-seq in S. coelicolor using 3×Flag-tagged SvCutRS (Fig. 1) and identified a core regulon of 16 CutR targets that are bound by both ScCutR and SvCutR in S. coelicolor M145 (Fig. 2, Table 6).
These core targets include the cutRS promoter, the promoter upstream of the uncharacterised ECF RNA polymerase sigma factor gene sco5147, and eight genes encoding proteins with putative cell envelope functions, including sco3977 and sco4157, which encode the high-temperature requirement proteases HtrA3 and HtrB, respectively. HtrA-like proteins typically act as qualitycontrol proteases and chaperones to monitor secreted protein folding outside the cell and htrB is also regulated by the secretion stress sensing two-component system CssRS, which activates its expression and that of the other two conserved htrA-like genes in Streptomyces species, htrA1 and htrA2 [14]. CutR also binds upstream of sco1507, which encodes a putative vitamin K epoxide reductase (VKOR), a group of enzymes, which can replace DsbB-like enzymes and recycle the DsbA-family enzymes that catalyse disulphide bond formation in secreted proteins [15].

CutRS directly controls the production of HtrA3, HtrB and the putative VKOR enzyme SCO1507
The TMT proteomics data show that HtrA3 is 3.2-fold higher in wild-type S. coelicolor relative to ∆cutRS while HtrB is 12-fold higher in the ∆cutRS mutant relative to the wild-type strain ( Table 6). Together with the ChIP-seq data this indicates that CutR directly activates expression of htrA3 and directly represses expression of htrB in S. coelicolor. To further test this trend of CutRmediated repression and activation qRT-PCR was carried out and the results confirmed that CutR represses the expression of htrB and activates htrA3 (Fig. 3). The ChIP-seq and proteomics data also show that CutR directly activates production of the VKOR homologue SCO1507, which is 3.75-fold higher in wild-type S. coelicolor versus the ∆cutRS mutant (Table 5). VKOR proteins are involved in the formation of disulphide bonds in secreted proteins by recycling DsbA-like proteins in bacteria that lack DsbB [15]. Consistent with this, S. coelicolor encodes a DsbA homologue (SCO2634) but does not encode DsbB, suggesting DsbA might be recycled by VKOR SCO1507. Of the remaining 16 target genes pulled down by both Flag-tagged ScCutR and SvCutR in S. coelicolor, nine were either not detected in the proteomics experiment or were not significantly affected by the loss of CutRS, including CutRS which are not present in the ∆cutRS strain ( Table 5). The remaining four CutR target promoters are all activated by CutRS and drive expression of uncharacterized proteins of unknown function. These are the putative FxsA-family membrane protein SCO1422 (8.4-fold), the putative cold shock domain protein SCO4295 (3-fold), the ECF RNA polymerase sigma factor SCO5147 (2-fold) and its divergently encoded O-methyltransferase SCO5146 (2-fold), and the putative membrane protein SCO5530 (5.6-fold).

DISCUSSION
In this work, we confirmed that loss of CutRS increases the production of the redox active antibiotic actinorhodin in S. coelicolor and showed that production of the actinorhodin biosynthetic enzymes is significantly upregulated in a ∆cutRS mutant. This is glucose dependent with upregulation of the biosynthetic enzymes up to 10-fold on DNA agar with no glucose and up to 300fold on DNA agar with glucose (DNAD). This likely explains why the ∆cutRS mutant visibly over produces actinorhodin on DNAD but not DNA. However, we conclude that CutR-mediated repression of the actinorhodin BGC is indirect because CutR does not bind anywhere within this gene cluster. Instead, the majority of direct CutR targets are membrane or secreted proteins (Table 5 and S1). Furthermore, the combined ChIP-seq and proteomics data show that CutR directly controls, in an antagonistic way, the production of two quality-control proteases named HtrA3 and HtrB [14] that likely play a role in the secretion stress response. Another link to the secretion stress response is the enhanced production of the VKOR homologue SCO1507, which is predicted to act like DsbB and recycle DsbA after it has catalysed disulphide bond formation in secreted proteins [15]. Up to 75 % of Streptomyces secreted proteins have two or more cysteine residues, excluding those in lipoprotein signal peptides [25], and it has been proposed that these cysteines play a crucial role in protein folding (via disulphide bond formation) outside the cell. The link here with actinorhodin is tentative but intriguing. VKORs can use quinones as electron acceptors during disulphide bond formation [15]. As a benzoisochromanequinone homodimer, actinorhodin has the potential to act as an alternate electron acceptor for SCO1057 [26]. It is interesting to note that Lejeune et al. have proposed that the quinones in actinorhodin can capture excess electrons from reactive oxygen and nitrogen species during oxidative metabolism, thus acting as an anti-oxidant [27].
In summary, we propose that CutRS is likely involved in the secretion stress response and must work alongside the conserved CssRS two component system, which activates the expression of htrA1, htrA2 and htrB [14]. Understanding the interplay of   Fig. 3. qRT-PCR on htrA3 and htrB. Deletion of cutRS results in a 0.6-fold downregulation of htrA3 and 44-fold increase in htrB expression. Conversely htrA3 is upregulated in the wild-type strain in the presence of glucose (ninefold) and htrB is slightly downregulated (0.9-fold). These data follow the pattern of protein detection under the same conditions using TMT-proteomics. Error bars represent standard deviation across two-four biological and two technical replicates these systems, their opposing effects on HtrB production and the roles of the four conserved HtrA-like foldases will be crucial in understanding the bacterial secretion stress response in these bacteria. Furthermore, although the effects of cutRS deletion on antibiotic production are indirect, the fact that the cutRS mutant makes more actinorhodin in the presence of glucose could also be a useful industrial trait, allowing increased growth and antibiotic yields. Future work will focus on further understanding the interplay of glucose, CutRS activity and antibiotic production in Streptomyces species.